ABSTRACT: miRNA in CSF of CFS Exosomes are membrane-bound microvesicles (Figure 1) Figure 1. Exosome formation. that are released from cells and mediate cell-to-cell transfer of short (22-25 nucleotides), noncoding, message inhibitory RNAs (miRNAs), genomic DNA, proteins and membrane lipids. The exosome surface has CD9, CD63, CD81 and other ligands that bind to integrins and other receptors on endothelium and other target cells. The exosome and target cell membranes fuse and miRNAs are released into the cytoplasm. Proinflammatory roles include transport of transforming growth factor-, and secretion of nitric oxide, reactive oxidant species, IL-1 and IL-18. Exosomes may act as circulating hormone- or virus-like inflammasomes to transmit injury signals in organs such as the brain. Pre- and post- synaptic neurons and glia have active exosome systems that contribute to neural plasticity, but that also transport amyloid, tau and prion proteins and rabies viruses. miRNAs are products of short genes that are scattered throughout the genome. They bind to precise sequences in selected mRNAs. The binding prevents translation of the target mRNA and prevents protein synthesis. Over 1500 human miRNAs regulate about 50% of known proteins in cell-specific fashion. Exosome miRNAs may initiate, expand, and maintain dysfunctional phenotypes by preventing the expression of critical proteins in targeted cells. miRNAs may be biomarkers of aging, mild cognitive impairment, Alzheimer's, multiple sclerosis, cardiomyopathy, and other diseases. In human synapses, ten miRNAs regulate 80% of the 242 presynaptic and 304 post-synaptic proteins. Analysis of pooled specimens of cerebrospinal fluid by next generation RNA sequencing (NGS) identified 133 unique miRNAs. However, NGS is currently inefficient for studying the patterns of miRNAs from individual subjects. This is because the yield of miRNA is low so that >2 ml of cerebrospinal fluid per individual subject may be needed, or the samples pooled between subjects. Therefore, we propose to use quantitative PCR of miRNAs for this cerebrospinal fluid study. Exosome proteins include RNA-binding heat shock and other cytoplasmic proteins; cell surface receptors such as CD9, CD63, CD81 and HLA-G; and other transmembrane proteins. These may reveal the nature of the cell type that secreted the exosome. Preliminary Data. Chronic Fatigue Syndrome (CFS) and Gulf War Illness (GWI) veterans who also met CFS criteria have demonstrated significant, specific, central nervous system dysfunction. GWI subsets have been identified. miRNA contributes to these GWI subsets. This suggests objectively defined CFS phenotypes may also be discovered and discriminated by miRNA patterns (biosignatures). Exosome RNA, protein, and lipid components can be purified from fresh and -80C frozen CSF. NGS was performed on cerebrospinal fluid from sedentary control (SC) subjects at rest and two subsets of GWI subjects defined by their magnetic resonance imaging and responses to exercise. The 3 groups had unique miRNA patterns. MIR22 had equivalent levels in SC and the Stress Test Associated Reversible Tachycardia (START) and Stress Test Originated Phantom Perception (STOPP) specimens. Eleven miRNAs were found in only in SC. START had 7 unique miRNAs, and STOPP had 2 unique miRNAs. These patterns may provide insights into mechanisms of exercise-induced malaise, pain, autonomic and cognitive dysfunction as well as being phenotype - specific biomarkers. These data from GWI with CFS suggest phenotypes and specific miRNAs biomarkers in CFS. Objective. Like GWI, CFS is a heterogenous disorder with latent phenotypes that can be identified from specific combinations of miRNAs expressed in cerebrospinal fluid exosomes. This unique hypothesis presumes that subsets of CFS have specific patterns of brain cell miRNAs. Distinct patterns of exosome miRNA and protein expression will discern CFS subjects from SC, and provide biomarkers for diagnosis. Exosome proteins may identify presynaptic, postsynaptic, microglial, astrocyte, or oligodendrocyte cells of origin. Drugs that target exosome biology may be beneficial in CFS or selected CFS phenotypes. SPECIFIC AIM 1: Isolate cerebrospinal fluid exosomes. Use quantitative polymerase chain reaction (Q- PCR) to identify the patterns of 380 miRNAs expressed in individual CFS and SC subjects before and after exercise. Determine patterns that discriminate CFS from SC, and identify latent phenotypes of CFS subjects. Characterize the miRNA-regulated proteins and infer the consequences for CFS brain biology. SPECIFIC AIM 2: Isolate cerebrospinal fluid exosomes and purify their proteins. Perform mass spectrometry for proteomic identification of exosome proteins. Determine the potential sources of the exosomes based on these proteins, and potential target cells based on the nature of the exosome surface receptors, transmembrane and intravesicular proteins.